System and method for metered dosage illumination in a bioanalysis or other system

ABSTRACT

A system and method for metered dosage illumination in a bioanalysis or other system includes an illumination system or subsystem that can provide optimized amounts of excitation light within the short exposure times necessary to measure fast biological activity. The amount of light can be precisely measured to provide quantitative results. The light flux can be precisely controlled to generate a prescribed minimum amount of light, in order to reduce adverse lighting effects on both fluors and samples. The system and method is particularly useful in any quality-control, analysis, or assessment-based environment. Typical research and development applications can include quality control, instrument calibration, and light output standardization; while clinical and diagnostics applications can include clinical monitoring, bioassay calibration and control for diagnostics, treatment and or therapeutic evaluation.

CLAIM OF PRIORITY

This application is a continuation-in-part of U.S. Patent Applicationtitled “SYSTEM AND METHOD FOR METERED DOSAGE ILLUMINATION IN ABIOANALYSIS OR OTHER SYSTEM”, application Ser. No. 13/007,535, filedJan. 14, 2011, which is incorporated herein by reference.

COPYRIGHT NOTICE

A portion of the disclosure of this patent document contains materialwhich is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure, as it appears in the Patent and TrademarkOffice patent file or records, but otherwise reserves all copyrightrights whatsoever.

FIELD OF INVENTION

The invention is generally related to light sources, and bioanalytics orbioanalysis, and is particularly related to a system and method formetered dosage illumination in a bioanalysis or other system, includingapplications in research and development; and in clinical and diagnosticarenas.

BACKGROUND

Bioanalytics or bioanalysis is the analysis of biological samples.Bioanalysis systems often use light to excite fluorescence frommolecular tags in a sample (referred to herein as fluorescent tags orfluors). Fluors may be exogenous, as in the case of fluorescentlylabeled immunochemical tags that recognize subcellular structure andbind to impose fluorescent labeling; or endogenous, as in the case ofgenetically modified cells in which fluorescent proteins for example areexpressed in order to impart specific fluorescent signals within aliving cell. Particularly in fields such as fluorescence imaging, geneexpression analysis, various types of sequencing, high resolutionfluorescence microscopy, fluorescence life time measurements, and highcontent screening, it is necessary to be able to measure the amount offluorescence and compare that amount to other measurements. This meansthe excitation light flux must generally be measured for eachillumination exposure or fluorescence excitation event. To monitorbiological events, it is necessary to be able to measure specificbiological activity with often sub-millisecond exposure times.Excitation and detection must occur rapidly, such that the illuminationmust be generally switched on and off within a time period that is atmost one-tenth of the duration of the minimum exposure time.Additionally, fluors can be temporarily or permanently photo-bleached,and biological samples can be otherwise damaged by the illuminationlight—a process known as phototoxicity. In order to minimizeperturbation of the fluors due to photo-bleaching, and photo-damage tothe biological samples, it is generally desired to minimize the lightflux or dosage within the constraints of the signal-to-noise (S/N)requirements of that particular bioanalysis system. These are thegeneral areas that embodiments of the invention are intended to address.

SUMMARY

Described herein is a system and method for metered dosage illuminationin a bioanalysis or other system. In accordance with an embodiment, anillumination system or subsystem is described that can provide optimizedamounts of excitation light within the short exposure times necessary tomeasure fast biological activity. The amount of light can be preciselymeasured to provide quantitative results. The light flux can also beprecisely controlled to generate only a prescribed minimum amount oflight, in order to reduce adverse lighting effects on both fluors andsamples. Although the examples herein illustrate the providing ofmetered dosage illumination in the context of a bioanalysis system, thetechniques can be similarly used to provide metered dosage illuminationin the context of other types of system. In accordance with variousembodiments, the technique is particularly useful in anyquality-control, analysis, or assessment-based environment. Typicalresearch and development applications can include quality control,instrument calibration, and light output standardization; while clinicaland diagnostics applications can include clinical monitoring, bioassaycalibration and control for diagnostics, treatment and or therapeuticevaluation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a system for metered dosage illumination, inaccordance with an embodiment.

FIG. 2 illustrates an example of a graphical user interface (GUI), foruse with a system for metered dosage illumination, in accordance with anembodiment.

FIG. 3 illustrates an example of an alternative graphical user interface(GUI), for use with a system for metered dosage illumination, inaccordance with an embodiment.

FIG. 4 illustrates an example of a fluorescence lifetime imagingmicroscopy (FLIM) technique, in accordance with an embodiment.

FIG. 5 illustrates an embodiment of an analog measurement of dosage, inaccordance with an embodiment.

FIG. 6 illustrates how the system can be used to provide constantcurrent dosage illumination, in accordance with an embodiment.

FIG. 7 illustrates how the system can be used to provide metered dosageillumination, in accordance with an embodiment.

FIG. 8 illustrates how the system can be used to provide constantintensity illumination, in accordance with an embodiment.

DETAILED DESCRIPTION

As described above, in the context of bioanalysis, it is often requiredthat an excitation light flux be measured for each illumination exposureor fluorescence excitation event; or that the illumination be switchedon and off within an appropriately brief time period; or that the lightflux or dosage be minimized within the constraints of thesignal-to-noise (S/N) requirements of the particular bioanalysis system.

To address this, described herein is a system and method for metereddosage illumination in a bioanalysis or other system. In accordance withan embodiment, an illumination system or subsystem is described that canprovide optimized amounts of excitation light within the short exposuretimes necessary to measure fast biological activity. The amount of lightcan be precisely measured to provide quantitative results. The lightflux can also be precisely controlled to generate only a prescribedminimum amount of light, in order to reduce adverse lighting effects onboth fluors and samples. In accordance with various embodiments, thetechnique is particularly useful in any quality-control, analysis, orassessment-based environment. Typical research and developmentapplications can include quality control, instrument calibration, andlight output standardization; while clinical and diagnosticsapplications can include clinical monitoring, bioassay calibration andcontrol for diagnostics, treatment and or therapeutic evaluation.

FIG. 1 illustrates a system for metered dosage illumination, inaccordance with an embodiment. As shown in FIG. 1, in accordance with anembodiment, the system 102 comprises a multi-color light engine 104 thatcan provide a plurality of different colors of light as a light output106, to excite various fluorescent markers in a sample 108. An exampleof such a light engine can be the SPECTRA series of light enginessupplied by Lumencor, Inc.

A host computer 110 generates an analysis protocol, including providinga dosage for each of the plurality of colors, and receiving feedbackdata from a camera 112. In accordance with an embodiment, the dosage canbe in the form of a reference count that represents the amount of lightflux per exposure period for each color provided by the light engine.

The camera is configured to detect fluorescence from the sample.

A beamsplitter 114 directs a portion of the light engine output, fromthe light engine, to a photodiode detector 116. A light-to-frequencyconverter 118 converts the detector output to a pulse train 130, thefrequency of which is proportional to the light flux. Amicroprocessor-based counter 120 counts the pulse train, for each color,and compares the ongoing count to the reference count or preset dosagefor each color.

Depending on the particular implementation, the above components can beprovided as part of a complete system; or alternatively some or all ofthe above components can be provided as part of an illuminationsubsystem. For example, in accordance with an embodiment, themulti-color light engine, beamsplitter, photodiode detector,light-to-frequency converter, and microprocessor-based countercomponents can be provided as an illumination subsystem intended for usewith a separately-provided host computer and camera.

In accordance with an embodiment, during operation of the system, thehost computer downloads a reference count 122 for each color to themicroprocessor-based counter. For each exposure period, the computertriggers the camera to begin an exposure period 124. The camera thensends an enable message or otherwise enables 126 the light engine, toturn on the appropriate color, and begins integrating the fluorescence.A portion of the light engine output 128, as directed by thebeamsplitter, is monitored by the photodiode detector. The detector'soutput is converted to the pulse train that is then counted by thecounter. When the counter reaches the reference count, it disables 132the color channel currently on or provided by the light engine.Simultaneously, another trigger 134 is sent to the camera, ending thecurrent exposure period.

The above approach avoids the latency inherent in any non-real-timeoperating systems. In accordance with an embodiment, additionalcircuitry can be provided to determine which color should be activated,and eliminate any delay inherent in the light engine turning on a colorchannel. An alternative approach is for the computer to turn on eachcolor directly in conjunction with starting the camera's exposureperiod. In yet other embodiments, the procedure described above can bemodified to suit the particular needs of the system (such as its use inquantitative analyses, or temporally fast analyses, as described below).

FIG. 2 illustrates an example of a graphical user interface (GUI) 142,for use with a system for metered dosage illumination, in accordancewith an embodiment. As shown in the example of FIG. 2, the GUI allows anoperator to set and to monitor dosage counts in each of the plurality ofcolors that can be provided by the light engine.

FIG. 3 illustrates an example of an alternative graphical user interface(GUI) 144, for use with a system for metered dosage illumination, inaccordance with an embodiment. In the example shown in FIG. 3, the GUIcontrols power levels and exposure times. For each color channel, theoperator can enter the relative power levels, maximum dosage frequencyand approximate exposure period. In accordance with an embodiment, thesystem, or software therein, calculates the required dosage or referencecount by multiplying the frequency by the desired exposure time. Theexposure time is approximate, since the intensity can change resultingin shorter or longer exposure periods, but still yielding the samemetered dosage for each period. If the power level is reduced (forexample, by half) the dosimeter frequency is similarly reduced by halfassuming a linear response. For the same exposure period the calculateddosage is reduced by half.

Quantitative Analysis

In accordance with an embodiment of particular use in quantitivateanalysis, the system can comprise a collection of color channels,wherein each color channel is used to provide the specific wavelengthband needed to excite a specific fluorescence molecule as defined by thefluorescence absorption of the tag. In accordance with an embodiment,each color channel can comprise a solid state light source (e.g. an LED,laser, light pipe or other light source), a single band pass filter, andan electronic circuit to power the light source. Within the system, thechannels can be combined using, e.g. dichroic mirrors, so that the lightfrom each source travels the same optical path, which is required, e.g.in epifluorescence measurements.

In accordance with an embodiment, the control circuitry can use aphotodiode to measure the excitation light flux from each channel duringeach exposure period. An example of such a photodiode can be the TAOSlight-to-frequency converter (part number TSL230RD), which can be usedto quantify the light flux or dosage for each illumination pulse. Theconverter circuit generates pulses at a rate that is proportional to thelight flux. Counting the pulses yields a measure of the dosage duringeach exposure time. Since dosage measurements can be made for each colorchannel independently, the user can optimize the amount of light, usinga GUI such as that described above, to irradiate any given fluoruniquely. This allows for differences in efficiencies of all theprocesses involved in fluorescent signal generation to be taken intoaccount for each color channel, (e.g. efficiency of light generation,fluorescence generation, or various differences in biological samplecondition and fluor concentration).

In accordance with an embodiment, quantitative fluorescent measurementscan be obtained ratiometrically by measuring the fluorescent signal, anddividing that signal by the dosage. In this manner, the fluorescentmeasurements can be normalized and rendered independent of theillumination intensity. This approach can be further automated by usingdosage measurements to control the timing of the illumination anddetection events. For example in accordance with an embodiment, adesired dosage count for each color channel can be entered into areference counter. During a measurement, the control electronics monitorthe dosage and gate off the excitation source for each color when thedosage count equals the amount in the reference counter. Light ismetered independently of the exposure time period, rather as a functionof the total light delivery prescribed by the reference counter. Theprecise dosage is delivered during each exposure, leading toquantitation as precise as the light flux measurement.

In this way, quantitative analysis can be performed using metered dosagewith or without constant exposure times. In the former case, the lightflux is maintained at the same level, and the flux is delivered withinthe exposure period. After the metered light dosage has been delivered,the excitation light is gated off leaving a time period when the cameracan still detect fluorescent signals. In the latter case, the light fluxis held constant, and the exposure time will vary as the intensityfluctuates.

In accordance with an embodiment, a range of light levels can bemonitored and measured using conventional counters and logic circuitry.In some instances, the dosage levels may be so small as to generateinsufficient statistical counts or too large and can overflow thecounters. In these instances, a programmable gain can be used todynamically adjust the count rate from the dosimeter circuitry.

Temporally Fast Analysis

In accordance with an embodiment of particular use in temporally fastanalysis, the sources and circuitry can be implemented for fastswitching, as is common with LEDs, lasers and light pipes. For example,in accordance with these embodiments, all switching can be performedelectronically, with no mechanical motion. This allows modulation in therange of 10 ns to 1 ms to be readily achieved. In addition, significantspeed increases can be achieved by directly connecting the camera andthe illumination subsystem. In accordance with this embodiment, the hostcomputer can trigger the camera to begin the analysis. The camera inturn can send a trigger to the illumination subsystem to turn on theexcitation light. When the dosage count equals the reference count, thelight is gated off, and a signal is sent to the camera to end theexposure. This configuration eliminates the variability in timingassociated with non-real-time operating systems.

The illumination subsystem can also be used as a direct connection todrive the camera to activate or advance. Color channels can beprogrammed, e.g. via a ring buffer, to engage camera operation uniquelyfor each fluor of interest, each excitation source in a preprogrammedseries of exposures of the various color channels. The timing of suchcamera and/or illuminator pulses can be optimized to interrogatespecialized fluor characteristics, such as for fluorophor lifetimemeasurements, photoactivation and photolysis measurements as examples.

Photo-Bleaching and Phototoxicity

In accordance with another embodiment, the ability to precisely meterthe light dosage and precisely turn on and off the illumination canminimize overall the exposure of the biological sample to theilluminator's excitation light. In so doing, photo-bleaching andphototoxicity effects are reduced, sample viability for live cellanalyses is prolonged, and artifacts imposed by the lighting on themeasurement are reduced. The combined benefit of such dosageoptimization is enhanced accuracy and longer duration quantitativefluorescent analyses.

Modulation Techniques

As described above, in accordance with an embodiment, a metered dosagetechnique can be used to obtain pulse-to-pulse repeatability andincrease dynamic range. This technique involves gating the light on andoff for the required dosage during each camera exposure period. Othermodulation techniques can be used, e.g., Förster (or Fluorescence)Resonance Energy Transfer (FRET); or fluorescence lifetime imagingmicroscopy (FLIM), wherein the illumination is modulated, and thefluorescence lifetime is determined by the phase shift and modulationdepth of the fluorescence signal relative to the illumination signal.FIG. 4 illustrates an example of a fluorescence lifetime imagingmicroscopy (FLIM) technique 146, in accordance with an embodiment.

Analog Measurement Techniques

As described above, in accordance with an embodiment, a host computercan be used to generate an analysis protocol, including providing adosage for each of the plurality of colors, and receiving feedback datafrom a camera, while a microprocessor-based counter counts the pulsetrain, and compares the ongoing count to the reference count or presetdosage for each color. in accordance with other embodiments, alternativemeasurement techniques, such as analog measurement techniques can beused. FIG. 5 illustrates an embodiment 152 which provides an analogmeasurement of dosage, in accordance with an embodiment. As shown inFIG. 5, the current output of a photodiode (PD) 154 can be fed to atransimpedance amplifier (TIA) 156, and converted to a voltage. Thisvoltage signal can be integrated by the integrator opamp (∫) 158. Thecomparator (=) 160 compares the integrator output to a target referencelevel equal to the desired dosage. When the two values are the same, thecomparator can generate a signal 162, turning off the light engine.

Use Cases

In accordance with an embodiment, the light engine can be operated indifferent modes, i.e. the output of the light engine can be controlledusing a variety of different mechanisms, to satisfy particular usecases. This enables the light engine to be particularly useful in, e.g.quality-control, analysis, or assessment-based environments.

For example, although some light engines might provide an open loopoption, in which output power information is readable on a monitor, suchinformation is generally not fed back to any controller internal to thelight engine. Although the current may be constant, there is no feedbackmechanism on the power delivered, so the outputs are susceptible to avariety of atmospheric changes. In accordance with an embodiment, theuse of a closed-loop option is beneficial not only for controllinginter-assay reproducibility, but also for monitoring inter-instrumentreproducibility. For example, using a metered dosage approach, an entitycan calibrate all of their light engines for all instrument products todeliver the same output power at a particular setting.

As another example, in analytical and clinical testing facilities,applications such as bioassay development, quality control in assaydevelopment and reagent formulation, clinical screening, diagnostics,treatment and therapeutic evaluation, and other analytical andbioanalytical endeavors, require that intra-assay control, as well asinter-instrument control, be robust and reproducible. This ensures thatmultiple instruments (e.g., readers, scanners and hand held analysistools) processing the same or similar analyses will yield equivalentresults in terms of the signal generated for similarly performedprocedures. In turn, this requires the instrument performance itself tobe well behaved and reproducible, which can only be accomplished inlight-dependent bioanalytical equipment if the illumination can becontrolled and identical for all instruments. In accordance with anembodiment, a metered dosage approach can be used to enable this type ofquality control. In particular, the output of each instrument can becalibrated to, e.g. a NIST traceable standard, and the dosage requiredto achieve this power level can be recorded. Then, this identical lightflux dosage, or an amount proportional to this dosage, can be deliveredfor each exposure for each instrument, or among different instrumentswithin a family of analysis tools.

Use Case—Constant Current

In accordance with an embodiment, in a constant current mode, the lightengine's circuitry monitors the current through each light source andkeeps that constant. The amount of current can be sensed by measuring avoltage drop across a calibrated resistor. Once thermalized, the poweroutput is constant for a constant ambient temperature and pressure, andthe pulse-to-pulse reproducibility is better than 99%. This is asuitable option in a well-controlled environment with no thermal changesor warm up times. However, outputs can fluctuate as a function of warmup, duty cycle, power level and atmospheric conditions.

FIG. 6 illustrates how the system can be used to provide constantcurrent dosage illumination, in accordance with an embodiment. As shownin FIG. 6, the system 170 can be provided similarly to that shown inFIG. 1 above. When a constant current mode is used, the light engine'scurrent monitoring circuit 172 monitors the current through each lightsource and keeps that constant, to provide a constant current lightoutput 176 of generally constant dosage 176 (subject to theconsiderations of warm up, duty cycle, power level and atmosphericconditions, as described above).

Use Case—Metered Dosage

In accordance with an embodiment, in a metered dosage mode, the systemmonitors a portion of the light engine output beam, internal to thelight engine, using a light-to-frequency converter or circuitry, similarto that described above. The output of this circuitry is a pulse trainwhich is fed to a counter. When the counter reaches the desired count ordosage, the light exposure is terminated. The total integrated lightflux or dosage can be regulated to be the same for each camera exposureor defined detection period. This means that, although the time periodthat the light source is on may vary slightly, the number of counts doesnot. The output can be locked into the photon number, and the resultantdosage is not susceptible to warm up time, duty cycle, power level orchanges in atmospheric conditions.

FIG. 7 illustrates how the system can be used to provide metered dosageillumination, in accordance with an embodiment. As shown in FIG. 7, thesystem 180 can again be provided similarly to that shown in FIG. 1above. When a metered dosage mode is used, the system monitors a portionof the light engine output beam, internal to the light engine, using alight-to-frequency converter or circuitry, and regulates the lightoutput 182 to be the same count 184, 186, 189 for each camera exposureor defined detection period, so that, although the time period that thelight source is on may vary slightly, the number of counts does not.

Use Case—Constant Intensity

In accordance with an embodiment, in a constant intensity mode, thelight engine can include a circuitry that monitors the light output, andadjusts the intensity levels in real time. This mode is particularlyuseful in rolling shutter camera applications. Limitations of thisapproach versus, e.g. the metered dosage approach described above,include that there may be a compromise in the maximum power or intensitydeliverable because some headroom is needed in order to offset theanticipated fluctuations; exposure times will generally be longer; andthe dynamic range of the final instrument will be smaller than that foran instrument controlled by metered dosage. The accuracy of the lightengine calibration will also be somewhat lower than that for metereddosage, because the sensor response will fluctuate due to atmosphericconditions.

FIG. 8 illustrates how the system can be used to provide constantintensity illumination, in accordance with an embodiment. As shown inFIG. 8, the system 190 can again be provided similarly to that shown inFIG. 1 above. When a constant intensity mode is used, a light intensitymonitoring/adjusting circuit 192 monitors the light output 194, andadjusts the intensity levels of one or more sources 196, 197 in realtime.

The present invention may be conveniently implemented using one or moreconventional general purpose or specialized digital computers ormicroprocessors programmed according to the teachings of the presentdisclosure. Appropriate software coding can readily be prepared byskilled programmers based on the teachings of the present disclosure, aswill be apparent to those skilled in the software art.

In some embodiments, the present invention includes a computer programproduct which is a storage medium (media) having instructions storedthereon/in which can be used to program a computer to perform any of theprocesses of the present invention. The storage medium can include, butis not limited to, any type of disk including floppy disks, opticaldiscs, DVD, CD-ROMs, microdrive, and magneto-optical disks, ROMs, RAMs,EPROMs, EEPROMs, DRAMs, VRAMs, flash memory devices, magnetic or opticalcards, nanosystems (including molecular memory ICs), or any type ofmedia or device suitable for storing instructions and/or data.

The foregoing description of the present invention has been provided forthe purposes of illustration and description. It is not intended to beexhaustive or to limit the invention to the precise forms disclosed. Inparticular, although most of the examples above illustrate the providingof metered dosage illumination in the context of a bioanalysis system,the techniques can be similarly used to provide metered dosageillumination in the context of other types of systems. The embodimentswere chosen and described in order to best explain the principles of theinvention and its practical application, thereby enabling others skilledin the art to understand the invention for various embodiments and withvarious modifications that are suited to the particular usecontemplated. It is intended that the scope of the invention be definedby the following claims and their equivalence.

What is claimed is:
 1. A system for providing metered dosageillumination for use in a bioanalysis or other system, comprising: alight engine that can provide a plurality of different colors of lightas a light output; a light detector that can detect an indication oflight flux at the light output wherein the light detector monitors aportion of the light output, internal to the light engine; alight-to-frequency converter that can convert the indication of lightflux as detected by the light detector to a pulse train, the frequencyof which pulse train is proportional to the light flux; a counter thatcan compare an ongoing count as indicated by the pulse train, to apreset reference count for each of the plurality of different colors oflight; and wherein, during operation, the system determines a referencecount for each color of the plurality of different colors of light, foran exposure period, enables the light engine to turn on a particularcolor of the plurality of different colors of light, monitors theindication of light flux in the light output, as detected by the lightdetector and converted to the pulse train counted by the counter for theparticular color of the one or more of the plurality of different colorsof light, and when the ongoing count reaches the reference count for theparticular color of the one or more of the plurality of different colorsof light, causes the light engine to turn off said particular color. 2.The system of claim 1, further comprising a fluorescent light detectorfor measuring fluorescence from a sample in response to the light outputfrom the light engine; wherein the fluorescent light detector gates onwhen the light engine turns on said one or more of the plurality ofdifferent colors of light; and wherein the fluorescent light detector isgated off when the ongoing count reaches the reference count for all ofthe one or more of the plurality of different colors of light.
 3. Thesystem of claim 1, wherein the system triggers on a camera forquantitative measurements and gates the camera off when the ongoingcount reaches the reference count for each of the one or more of theplurality of different colors of light.
 4. The system of claim 1,wherein the system further comprises a camera for measuringfluorescence, wherein the system triggers the camera to begin anexposure period when the light engine turns on one of the plurality ofdifferent colors of light; and wherein the system triggers the camera toend the exposure period when the ongoing count reaches the referencecount for each of the plurality of different colors of light.
 5. Thesystem of claim 1, wherein the system further comprises a camera forimaging and wherein the system triggers the camera to begin an exposureperiod when the light engine turns on one of the plurality of differentcolors of light; and wherein the system triggers the camera to end theexposure period when the ongoing count reaches the reference count foreach of the plurality of different colors of light.
 6. The system ofclaim 1, wherein the system is used as part of a bioanalysis system. 7.The system of claim 1 wherein the system is used as part of abioanalysis system which, is used for detection of fluorescent tags orfluors in a biological sample.
 8. The system of claim 1, wherein thesystem is used as part of a bioanalysis system which is used for one ormore of: quantitative analyses, temporally fast analyses, fluorescenceimaging, gene expression analysis, sequencing, high resolutionfluorescence microscopy, fluorescence life time measurements, ForsterResonance Energy Transfer (FRET) Microscopy, and high content screening.9. The system of claim 1, wherein the light engine is configured tooperate in a constant current mode, wherein a current monitoring circuitmonitors the current through one or more light source and keeps thecurrent constant, through said one of more light source, to provide aconstant current light output of generally constant dosage.
 10. Thesystem of claim 1, wherein the light engine is configured to operate ina metered dosage mode, so that, although the time period that the lightengine is on may vary slightly, the number of counts does not.
 11. Amethod for providing metered dosage illumination for use in abioanalysis or other system, comprising the steps of: providing a lightengine that can provide a plurality of different colors of light as alight output; providing a light detector internal to the light enginethat can detect an indication of light flux in the light output for eachof said plurality of colors; providing a light-to-frequency converterthat can convert the indication of light flux as detected by the lightdetector to a pulse train, the frequency of which pulse train isproportional to the light flux; providing a counter that can compare anongoing count as indicated by the pulse train, to a preset referencecount for each of said plurality of colors; and during operation of theillumination system determining a reference count for each of saidplurality of colors, for an exposure period, enabling the light engineto turn on a particular color of said plurality of colors, monitoringthe indication of light flux at the light output, as detected by thelight detector and converted to the pulse train counted by the counterfor the particular color of said plurality of colors, and when theongoing count reaches the reference count for the particular color ofsaid plurality of colors, turning off the particular color.